PROJECT SUMMARY The evolutionarily-conserved Wnt signal transduction pathway directs cell proliferation and differentiation during animal development and tissue homeostasis. Despite the fact that deregulation of Wnt signaling underlies numerous developmental disorders and cancers, including nearly all colorectal cancers, many of these mechanisms remain poorly understood. Thus, a deeper understanding of the mechanisms that activate this pathway will guide the development of new therapeutic strategies to combat Wnt-driven diseases. The long-term goal of the PI?s research program is to elucidate the mechanisms that activate Wnt signaling during animal development and to use this knowledge to identify control points in the pathway susceptible to therapeutic targeting in Wnt-driven diseases. In support of this effort, the PI and her laboratory group have established innovative in vivo models in Drosophila that have revealed unanticipated functions of three core Wnt pathway effectors: the tumor suppressor, APC; the scaffold protein, Axin; and the ADP-ribose polymerase, Tankyrase. Building on these findings and enabled by genome-wide screens designed to uncover new Wnt signaling regulators, the current project will address three major unsolved questions: 1) how the membrane- associated receptor activation complex is assembled and activated; 2) how the negative regulatory cytosolic beta-catenin destruction complex is inhibited upon Wnt stimulation; and 3) how the nuclear beta-catenin-TCF transcription complex is activated. To elucidate the role of previously unknown Wnt pathway activators, this project will couple genetic, cell biological and biochemical approaches with in vivo assays previously developed by the PI to monitor pathway activation within Wnt signaling gradients. It will apply an innovative focus centered on three components: a deubiquitinating complex essential for Wnt receptor stability, a ubiquitin ligase essential for signaling activation, and a kinase that activates the beta-catenin-TCF transcription complex. This work is complemented by productive collaborations with investigators who have expertise in the biochemical reconstitution of Wnt signaling, Wnt pathway analysis in vertebrate models, and in the identification of Wnt-dependent post-translational protein modifications using mass spectrometry analysis. The successful completion of this work will provide an understanding of: 1) the control of Wnt receptor activity; 2) the control of the beta-catenin destruction complex; 3) the activation of the Wnt transcription complex; and 4) novel therapeutic strategies to target Wnt-driven diseases.